We are continuing the molecular cloning and characterization of a novel gene that is expressed in cytotoxic mouse macrophages. The gene was obtained by screening a mouse macrophage cDNA library with a cDNA probe containing sequences found in several genes including the mouse IL-2 gene. Nucleic acid sequencing revealed that the cDNA represents a unique gene and that it encodes a predicted protein of at least 21 kDa. The gene contains a region of > 100 bp that encodes a stretch of glutamines followed by glutamines that are interspersed with histidine. Overall, the gene is expected to encode a highly hydrophilic, basic protein. Northern blotting and reverse transcriptase-polymerase chain reaction analyses indicated that the gene is express constitutively in multiple cell types. Based on the above characteristics and partial nucleic acid sequence similarity to other known genes, this gene may encode a protein that functions as a DNA-binding transcription factor or a protein involved in cell- or tissue-specific differentiation or maturation. We have continued to study cytokine regulation of nitric oxide (NO) synthesis by macrophage. Th1 and Th2 cell clones were observed to differentially regulate macrophage NO synthesis via the co-stimulatory actions of IFN-gamma and IL-2 and the inhibitory action of IL-4, respectively. Picolinic acid, previously shown to be a costimulator of NO production, was found to act with IFN-gamma as transcriptional activator of NO synthase. MSP-1 was found to be a potent inhibitor of endotoxin and cytokine-induced NO synthesis by macrophage. Newly initiated studies have addressed the possibility that NO may function as autocrine/paracrine regulator of macrophage and other cells. NO-releasing agents were found to function in a dose-dependent, biphasic manner to increase and decrease IFN-gamma plus LPS-induced NO synthase gene and protein expression in macrophage.